It is generally known that quantitative analysis of creatine kinase (CK or CPK) in blood sampled from a patient is diagnostic of creeping palsy, dermatomyositis and cardiac infarction. In creatine kinase (CK), there are three isozymes, namely, creatine kinase MM (CKMM), creatine kinase MB (CKMB) and creatine kinase BB (CKBB). These isozymes are mainly present in skeletal muscle (CKMM), cardiac muscle (CKMB), or brain and spinal cord (CKBB), respectively. When cardiac infarction occurs, CKMB in cardiac muscle comes out into blood and accordingly its content in blood increases. Therefore, it is of value for diagnosis of cardiac infarction to assay CKMB in blood.
Methods for assaying creatine kinase and/or creatine kinase MB isozyme had been studied, and a precise assaying method was established.
The method for assaying creatine kinase generally comprises the steps of preparing serum or plasma of the blood sampled from a patient, forming spectroscopically detectable species according to the enzyme activity of creatine kinase contained in the serum or plasma, and measuring the amount of the formed species to determine the content of creatine kinase.
For performing the above analytical method, the following two reaction systems are employable.
1) Reaction System 1
Creatine phosphate (CP) and adenosine diphosphate (ADP) are caused to react in the presence of creatine kinase (CK) contained in the sample (serum or plasma) to form adenosine triphosphate (ATP), while pH condition of the enzyme reaction is adjusted with a buffer working in the pH range of 5.5 to 8.5. The amount of the formed adenosine triphosphate (ATP) is proportional to that of creatine kinase (CK). The thus formed ATP is caused to react with glucose (Glu) in the presence of hexokinase (HK) to prepare glucose-6-phosphate (G6P), and then the prepared G6P is caused to react with nicotinamide adenine dinucleotide(phosphate) in an oxidized form (NAD(P)) in the presence of glucose-6-phosphate dehydrogenase (G6PDH) to produce nicotinamide adenine dinucleotide(phosphate) in a reduced form (NAD(P)H). The amount of the produced NAD(P)H is measured by a spectroscopic method and determined in accordance with the calibration curve beforehand obtained. The content of creatine kinase (CK) is determined from thus determined amount of NAD(P)H.
In order to improve accuracy of the spectroscopic measurement, NAD(P)H can be further caused to react with a tetrazolium salt to form a formazan dye and then the amount of the formazan dye is measured to determine the content of creatine kinase (CK) in the sample (Japanese Patent Provisional Publication No. 63(1988)-32499).
The reaction system 1 described above is represented by the following formulas.
______________________________________ CP + ADP .fwdarw. ATP + creatine (in the presence of CK) ATP + Glu .fwdarw. G6P + ADP (in the presence of HK) G6P + NAD(P) .fwdarw. NAD(P)H + 6-phosphogluconic acid (in the presence of G6PDH) NAD(P)H + tetrazolium salt .fwdarw. formazan dye + NAD(P) ______________________________________
2) Reaction System 2
Creatine phosphate (CP) and adenosine diphosphate (ADP) are caused to react in the presence of creatine kinase (CK) contained in the sample to form adenosine triphosphate (ATP), while pH condition of the enzyme reaction is adjusted with a buffer working in the pH range of 5.5 to 8.5. The amount of the formed adenosine triphosphate (ATP) is proportional to that of creatine kinase (CK). The thus formed ATP is caused to react with glycerol in the presence of glycerol kinase to prepare L-.alpha.-glycerophosphate, and then the prepared L-.alpha.-glycerophosphate is caused to react with oxygen in the presence of L-.alpha.-glycerophosphate oxidase to produce hydrogen peroxide. Finally, thus produced hydrogen peroxide is caused to react with a leuco dye to form a blue dye, and then the amount of the blue dye is measured by a spectroscopic method and determined in accordance with the calibration curve beforehand obtained. The content of creatine kinase (CK) is determined from thus determined amount of the blue dye.
The reaction system 2 described above is represented by the following formulas.
______________________________________ CP + ADP .fwdarw. ATP + creatine (in the presence of CK) ATP + glycerol .fwdarw. L-.alpha.-glycerophosphate + ADP (in the presence of glycerol kinase) L-.alpha.-glycerophosphate + O.sub.2 .fwdarw. H.sub.2 O.sub.2 + dihydroxyacetone phosphate (in the presence of L-.alpha.-glycerophosphate oxidase) H.sub.2 O.sub.2 + leuco dye .fwdarw. blue dye + H.sub.2 O ______________________________________
Since creatine kinase has the three isozymes described above, it is not easy to assay creatine kinase MB isozyme. Prior to assaying process, MB isozyme must be isolated from MM and BB isozymes in principle. Practically, the amount of BB isozyme in blood is negligible because it is essentially present in brain and spinal cord, but it is still very difficult to separate MB isozyme from MM isozyme.
For the purpose of obviating the above trouble, Japanese Patent Publication No. 56(1981)-19239 proposes an assaying method in which a particular antibody is employed, instead of isolating MM isozyme from the sample, to inhibit the activity of MM isozyme without adversely affecting that of MB isozyme. The antibody completely inhibits the enzyme activity of sub-unit M in MM and MB isozymes, but on the other hand it does not inhibit the activity of subunit B in MB isozyme. In the method, the sample (serum or plasma) is treated with the antibody (the M sub-unit inactivation antibody) so that creatine kinase in the sample may have only the activity originating from MB isozyme. Thereafter, thus restricted activity is measured by the above reaction system 1 or 2 to assay creatine kinase MB isozyme.
In the conventional assaying process for creatine kinase or creatine kinase MB isozyme, the reactions described above are generally performed in a solution (the process in which the reactions are performed in a solution is referred to as "wet process"). The wet process generally requires relatively long time, and accordingly it takes relatively long time to make a diagnosis based on the wet assaying process. However, for example in the case of cardiac infarction, it is needed to diagnose the case, as soon as possible, because the success of treatment depends on how soon the treatment begins. From this viewpoint, it is preferred to shorten the time to perform the assaying process.
For assaying process which can be performed in relatively short time, a dry analytical element is known. The analytical element generally comprises a transparent support and an agent layer provided thereon which contains a reagent composition participating in the reaction system for detection. For assaying the creatine kinase or creatine kinase MB isozyme, a dry analytical element is also developed and practically used. In the analytical element, the agent layer comprises the reagent composition for the above described reaction system 1 or 2. The detailed description about the dry analytical element for creatine kinase or creatine kinase MB isozyme is given in the following publications; Japanese Patent Provisional Publications No. 61(1986)-254198, No. 61(1986)-254199, No. 61(1986)-260164, and No. 63(1988)-32499.
Although the dry analytical element shortens the time to perform the assaying process, the reagent composition of the analytical element is liable to deteriorate during storage. If the analytical element is stored under ambient conditions, its sensitivity rapidly lowers. For obviating the deterioration of the reagent composition, the known dry analytical element must be stored in a refrigerator. However, it is very inconvenient to need a refrigerator for storing the analytical element, and further such inconvenience for storage often delays beginning of the assaying process and accordingly it often prevents an early definitive diagnosis.
Therefore, it is an object of the present invention to improve the storage stability of dry analytical element for quantitative analysis of creatine kinase or creatine kinase MB isozyme. Particularly, it is an object of the invention to provide a creatine kinase or creatine kinase MB isozyme dry analytical element which does not deteriorate even if stored under ambient conditions and hence which can be stored under ambient conditions, with no need of employing a refrigerator.